MiR-205-5p Functions as a Tumor Suppressor in Gastric Cancer Cells through Downregulating FAM84B

MicroRNAs (miRNAs) participate in the formation of multiple diseases, including gastric cancer (GC), through modulating specific targets. Here, we explored the functions and regulatory mechanisms of miR-205-5p in GC. MiR-205-5p levels were detected in GC cells through qRT-PCR. Besides, the role of miR-205-5p in cell proliferation, cell apoptosis, cell cycle, cell invasion, and metastasis was assessed through CCK-8 assay, colony formation, flow cytometry, scratch assay, transwell, and western blot. Moreover, the Starbase website was used to predict the target gene of miR-205-5p, further verified by a dual-luciferase reporter assay. Furthermore, the functional effects of the family with sequence similarity 84 member B (FAM84B) on GC mediated by miR-205-5p upregulation were further investigated. MiR-205-5p expression was decreased in GC cells. Upregulation of miR-205-5p inhibited cell proliferation and metastasis and induced apoptosis and cycle arrest of GC cells. Moreover, FAM84B was predicted and confirmed as a target of miR-205-5p and negatively related to miR-205-5p. Mechanically, FAM84B overexpression partially rescued the functional effects of miR-205-5p upregulation on GC cell progression. This study suggests the potential of miR-205-5p/FAM84B as novel targets for the treatment of GC.


Introduction
Gastric cancer (GC) is one of the most dangerous cancers worldwide with high morbidity and mortality [1]. Because of the great progress in the diagnosis and treatment, the longterm survival of GC patients has been remarkably prolonged in the past few decades. However, many patients have developed into advanced stages at the time of diagnosis due to lack of early symptoms. For GC patients with advanced stages of metastatic tumors, the therapeutic effects were disappointing with less than a 30% 5-year survival rate [2]. erefore, identifying effective early diagnostic biomarkers and treatment targets is the main task for GC control and prevention.
MicroRNAs (miRNAs) are nucleotides with a length of about 18-25 bp and do not encode proteins. MicroRNAs are closely involved in the regulation of posttranscriptional gene expression. Numerous evidence report that the expression profile of miRNAs was remarkably abnormal in tumors and played protumor or antitumor effects according to the function of their target genes. Recently, it was suggested that miRNAs were involved in the regulation of tumor cells' malignant phenotype, such as proliferation, apoptosis, differentiation, migration, and invasion. Several miRNAs have been implicated in GC progression, including miR-21-5p, miR-214, miR-143, miRNA-17-3p, and miRNA-17-5p [3][4][5][6]. For example, miR-21-5p promotes GC cells proliferation through modulating SMAD7 [3]. MiR-182 levels were reduced in GC patients. MiR-182 overexpression inhibited GC progression via targeting KLF4 [7]. Moreover, miR-205-5p was expressed at a low level in certain cancers, including renal carcinoma [8], prostatic carcinoma [9], breast cancer [10], colorectal cancer [11], and ovarian cancer [12]. MiR-205-5p inhibits angiogenesis in GC by attenuating the expression of VEGFA and FGF1 [13]. But the precise impacts of miR-205-5p on influencing GC progression and the related mechanisms remained largely unknown. is work focused on elucidating the biological functions of miR-205-5p on GC progression by examining its expression in GC cells and identifying its target.

qRT-PCR.
After extracting total RNA from cells, cDNA was prepared with the total RNA by the RNeasy plus micro kit through reverse transcription in line with specific instructions, as the starting material of qRT-PCR carried out using step one system (Life Technologies Corp). Sequences of all primers were designed by a Primer Premier software 4.0 (Premier, Canada) and as Table 1. U6 or GAPDH were normalized by the 2 −ΔΔCT approach [14].

Colony Formation Assay.
Transfected GC cells (1 × 10 3 / well) were cultured in 6-well plates, and the medium was replaced every 2-3 days for a total of two weeks. Later, GC cells were stained using 1% crystal violet for 30 min and imaged (Nikon, Japan).

Flow Cytometry.
To analyze cell apoptosis, the culture medium of GC cells from different groups was collected.
en Annexin V-FITC and PI were added for 10 min. Flow cytometry was conducted for measuring apoptotic cells. For the cell cycle, GC cells from different groups were harvested, followed by overnight fixation with 70% EtOH and 30 min of PI staining. Finally, flow cytometry was adopted for detecting cell cycle distribution.

Transwell
Assay. For invasion assays, Matrigel was dissolved with serum-free DMEM, and uniformly covered in the Transwell chamber for 1 h at 37°C. For migration and invasion assays, GC cells from different groups were seeded into upper chambers at the appropriate density. Medium containing 10% FBS was added into lower chambers. After 48-h incubation at a room temperature of 37 o C, migrated and invaded cells were stained with crystal violet (0.1%) and captured (Nikon, Japan) (200×). en, miR-205-5p mimic and pmirGLO-FAM84B WT/MUT or NC mimic were cotransfected for 48 h, and the dual-luciferase reporter system (Promega, USA) was utilized.

Statistical
Analysis. Data were analyzed using a GraphPad Prism 5.0 and presented as mean ± SD. Differences between the groups were compared by ANOVA as well as Tukey's poc host analysis. p < 0.05 was considered statistically significant.

MiR-205-5p Overexpression Suppressed GC Cells Proliferation and Metastasis.
QRT-PCR was used to examine the miR-205-5p expression in GC cells and it was found that miR-205-5p was expressed at a low level in GC cells, especially in AGS and SGC-7901 cells (Figure 1(a)). e miR-205-5p mimic was transfected into GC cells, and transfection efficiency is shown in Figure 1 en CCK-8 and colony formation were conducted for assessing the effect of miR-205-5p mimic on GC cells viability and proliferation. As shown in Figures 2(a) and 2(b), miR-205-5p upregulation decreased the viability and colony-forming ability of GC cells. In addition, flow cytometry was conducted for measuring cell apoptosis and cell cycle.

Data in Figures 2(c) and 2(d) indicated that miR-205-5p
overexpression promoted apoptosis and induced cell cycle arrest at the G0/G1 phase of GC cells. Furthermore, a western blot assay was employed for determining the effect of miR-205-5p mimic on cell cycle-related protein levels. Based on Figure 2(e), miR-205-5p overexpression decreased the protein expressions of cyclin D1 and CDK2 but increased that of P21.
Moreover, scratch and transwell analyses were carried out for assessing miR-205-5p overexpression on metastasis in GC cells. Figures 3(a) and 3(b) showed that miR-205-5p high-expression decreased the abilities of GC cell metastasis. Furthermore, the expression of metastasis-related proteins was also detected. As shown in Figure 3(c), miR-205-5p mimics repressed the expression of MMP2, MMP9, and COX-2.
ese data indicated that miR-205-5p overexpression repressed GC cells proliferation and metastasis.

MiR-205-5p Targeted FAM84B in GC.
MiRNAs regulated gene expression through binding to the 3′ UTR of target genes and inducing degradation of their mRNAs [16]. Bioinformatics tool (Starbase) was used to search for the possible miR-205-5p targets and FAM84B was found to be the most related gene. e dual-luciferase reporter analysis verified the target relationship (Figures 4(a) and 4(b)). Moreover, FAM84B levels in GC cells transfected with miR-205-5p mimic were detected through qRT-PCR and western blot. Data in Figures 4(c) and 4(d) showed that miR-205-5p overexpression caused FAM84B downregulation in GC cells. ese data indicated that FAM84B was the target of miR-205-5p in GC.

Discussion
Our data elucidated a novel molecular mechanism by which miR-205-5p acted as a tumor suppressor in GC cells through targeting FAM84B and inhibiting its expression. Evidences have revealed that miRNAs exert essential roles on GC tumorigenesis, development, and chemoresistance, and were associated with patients' characteristics and outcomes. For example, miRNA-194 was associated with good prognosis in GC patients and induced GC cell growth inhibition and cell cycle arrest [17]. Moreover, miR-3664-5p suppressed the proliferation and metastasis of GC by negatively modulating MTDH through attenuating NF-κB signaling pathway [18]. In addition, miRNA-192 and miR-215 enhanced the growth and migration of GC cells through targeting APC [19].   MiR-205-5p  5′-TCCTTCATTCCACCGGAGTCTG-3  5′-GCGAGCACAGAATTAATACGAC-3′  FAM84B  5′-GACCCACCTAAGTTACAAGGAAG-3′  5′-GTAGAACACGGAGCATTCCAC-3′  U6  5′-CTCGCTTCGGCAGCACA-3′  5′-AACGCTTCACGAATTTGCGT-3′  GAPDH 5′-CCTCGTCTCATAGACAAGATGGT-3′ 5′-GGGTAGAGTCATACTGGA ACATG-3′ Journal of Oncology 3 erefore. miRNAs were involved in multiple malignant phenotypes of GC through regulating differential gene expression. Here, we identified that miR-205-5p expression was decreased in GC cells. Particularly, miR-205-5p is expressed at low level and function as a tumor suppressor in multiple cancer types. Guo et al. found miR-205-5p was downregulated in gallbladder cancer (GBC). MiR-205-5p upregulation repressed drug resistance, proliferation, and promoted apoptosis of GBC stem cells [20]. In a recent study found that up-regulation of miRNA-205-5p exerted an antitumor effect accomplished by decreasing VEGFA and inactivating PI3K/Akt/mTOR in renal carcinoma cells [8]. However, in lung cancer, miRNA-205-5p was upregulated in tumor tissues and promoted cell proliferation and survival through modulating erbB3 [21]. e differential function of miRNA-205-5p might be due to the differences in the molecular microenvironment in cancer types. Since the targets of miRNAs varied in different cell types and disease contexts [22,23], it is necessary to identify the function of miRNA-205-5p on other cancer types.
MiRNA-205-5p upregulation suppressed the proliferation, metastasis, induced cell cycle arrest, and apoptosis of GC cells accompanied by decreasing cyclin D1, CDK2, and increasing P21. Cyclin D1, acts as a regulatory subunit of cyclin-dependent kinase (CDK4 or CDK6), which is necessary for G1 to S progression in the cell cycle [17]. Cyclin D1 is upregulated or mutated in various tumor types, which alters cell proliferation [24]. CDK2 is also a cell cycle Journal of Oncology 5 checkpoint protein, which controls G1 to S phase transition and forms complex with cyclin A or E [25,26]. P21 functions as a cyclin-dependent kinase inhibitor, which binds to CDK2 or CDK4 [27,28]. It is widely known that P21 was the important target protein of tumor suppressor p53, through which this protein mediated p53-dependent cell cycle arrest [29,30]. In summary, cyclin/CDK were the important downstream signal proteins in the antitumor function of miRNA-205-5p. Tumor metastasis, driven by cancer cell invasion and migration, was one of the most important causes for GC-related deaths [2]. Here, we demonstrated that miRNA-205-5p overexpression led to significant inhibition of GC cell metastasis by downregulating COX-2, MMP2, and MMP9 levels. COX-2, as a member of COX family, is closely related to tumorigenesis and invasiveness [31]. Matrix metalloproteinases (MMPs) are positive regulators of intestinal tumorigenesis [32]. FAM84B, known for its potential connections with Myc and Ras, is located at the chromosomal locus 8q24.21 [33]. e amplification of the 8q24.21 region had been found in various cancer types, including GC [34], ovarian cancer [35], prostate cancer [36], and others. Currently, the oncogenic Journal of Oncology function of Myc has been widely demonstrated, while few studies report similar results for FAM84B. Recently, the elevation of FAM84B expression has been reported in esophageal squamous cell carcinoma [37] and prostate cancer [38]. It is worth noting that FAM84B promoted tumor growth and metastasis in prostate cancer via activation of the AKT signal pathway [39]. However, whether FAM84B plays a role in GC progression remains unclear. We found that FAM84B was the target of miR-205-5p, and its overexpression could reverse the antitumor effect of miR-205-5p upregulation. Taken together, our study showed that miR-205-5p was decreased, and FAM84B increased in GC cells. Upregulation of miR-205-5p inhibited GC cell proliferation and metastasis, and promoted GC cell apoptosis and cell cycle arrest by regulating FAM84B. Our results suggested that miR-205-5p could be a potential target for the GC treatment.

Data Availability
All data generated or analyzed during this study are included in this published article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Xi Chen and Lei Zhang contributed equally to this work. Zhong Chen and XiaoPeng Cui conceived and designed the study. Xi Chen and Lei Zhang conducted most of the experiments. JingBo Geng analyzed the data and performed the literature search and data extraction. Xi Chen drafted the manuscript. Xi Chen and Lei Zhang finalized the manuscript. All the authors read and approved the final manuscript.